The Molecular Basis of Viral DNA Sensing through the cGAS-STING Pathway The innate immune response is the first line of defense against invading pathogens. Viral nucleic acids, such as DNA and RNA, serve as danger signals that induce potent immune responses in infected cells. The detection of viral nucleic acids is a central strategy by which the hosts sense infection and launch protective immune responses. Cyclic GMP-AMP synthase (cGAS) is a cytosolic dsDNA sensor in innate immunity. It is activated by viral DNA and catalyzes the synthesis of a noncanonical cyclic dinucleotide 2?, 3? cGAMP (referred to as cGAMP). As a second messenger, cGAMP stimulates the induction of type I interferons (IFNs) via the adaptor STING located on the ER membrane. The engagement of cGAMP by STING leads to the recruitment and activation of the protein kinase TBK1. The transcriptional factor IRF-3 is then recruited to the signaling complex by phosphorylated STING. The induced proximity of IRF-3 and TBK1 leads to IRF-3 phosphorylation and activation. Phosphorylated IRF-3 oligomerizes, translocates to the nucleus, and induces the expression of type I IFNs, conferring antiviral activity to the host and activating the acquired immune responses. To understand the molecular basis of viral DNA sensing through the cGAS-STING pathway, the PI determined the crystal structures of cGAS in isolation and in complex with dsDNA; the structures of STING in isolation and in complex with cGAMP; the structures of mouse TBK1 bound to two inhibitors; the structure of phosphorylated STING C-terminal peptide bound to IRF-3; and the structure of a phosphomimetic dimer of IRF-3. These comprehensive structural and functional studies significantly advanced our understanding about how the innate immune system responds to viral DNA at molecular level. However, it remains unknown how cGAMP binding activates STING and mediates the recruitment and activation of TBK1 and IRF-3 at the signaling complex. In addition, the exact mechanism of how phosphorylation induces the activation of IRF-3 and facilitates its association with the IFN-? promoter remains to be determined. Built-upon our strong preliminary studies, the proposed research will elucidate the molecular bases of several key steps of the cGAS-STING pathway with the following aims: 1). Determine the structural basis of TBK1 recruitment by STING; 2). Elucidate the molecular basis of STING activation by cGAMP; 3). Delineate the mechanism of IRF-3 activation by TBK1. The proposed studies represent a rigorous and comprehensive investigation into the mechanisms of cytosolic DNA sensing and will dramatically advance our understanding of the molecular basis of innate immunity against viral infection. In addition, they provide a structural framework for innovative approaches to treat viral diseases, autoimmune disorders, and cancer by manipulating the innate immune response.